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Purpose: We report neurocognitive, imaging, ophthalmologic, and safety outcomes following low-dose whole brain radiation therapy (LD-WBRT) for patients with early Alzheimer dementia (eAD) treated in a pilot trial., Methods and Materials: Trial-enrolled patients were at least 55 years of age, had eAD meeting NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association) Alzheimer's Criteria with confirmatory fluorodeoxyglucose and florbetapir positron emission tomography findings; had the capacity to complete neurocognitive function, psychological function, and quality-of-life assessments; had a Rosen modified Hachinski score ≤4; and had estimated survival >12 months., Results: Five patients were treated with LD-WBRT (2 Gy × 5 over 1 week; 3 female; mean age, 73.2 years [range, 69-77]). Four of 5 patients had improved (n = 3) or stable (n = 1) Mini-Mental State Examination (second edition) T-scores at 1 year. The posttreatment scores of all 3 patients who improved increased to the average range. There were additional findings of stability of naming and other cognitive skills as well as stability to possible improvement in imaging findings. No safety issues were encountered. The only side effect was temporary epilation with satisfactory hair regrowth., Conclusions: Our results from 5 patients with eAD treated with LD-WBRT (10 Gy in 5 fractions) demonstrate a positive safety profile and provide preliminary, hypothesis-generating data to suggest that this treatment stabilizes or improves cognition. These findings will require further evaluation in larger, definitive, randomized trials., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: To construct a tumor voxel dose response matrix (DRM) and dose prescription function (DPF) for adaptive dose painting by number (DPbN) based on treatment feedback of fluoro-2-deoxyglucose (FGD) positron emission tomography (PET)/computed tomography (CT) imaging., Methods and Materials: FDG-PET/CT images obtained before and after chemoradiation therapy and at weekly chemoradiation therapy sessions for each of 18 patients with head and neck cancer, as well as the treatment outcomes, were used in the modeling. All weekly and posttreatment PET/CT images were registered voxel-to-voxel to the corresponding pretreatment baseline PET/CT image. Tumor voxel DRM was created using serial FDG-PET imaging of each patient with respect to the baseline standardized uptake value (SUV 0 ). A tumor voxel control probability (TVCP) lookup table was created using the maximum likelihood estimation on the tumor voxel (SUV 0 , DRM) domain of all tumors. Tumor voxel DPF was created from the TVCP lookup table and used as the objective function for DPbN-based inverse planning optimization., Results: Large intertumoral and intratumoral variations on both tumor voxels (SUV 0 , DRM) were identified. Tumor voxel dose resistance did not show correlation with its baseline SUV 0 value and was the major cause of the tumor local failures. Tumor voxel DPF as the function of tumor voxel (SUV 0 , DRM) values also showed a very large intertumoral and intratumoral heterogeneity. Most human papillomavirus-negative tumors require a treatment dose >100 Gy to certain local tumor regions. These treatment doses, which are most unlikely to be implementable in conventional radiation therapy, can be achieved using adaptive DPbN treatment. Clinical feasibility was evaluated by comparing the adaptive DPbN treatment plan with the conventional intensity modulated radiation therapy plan., Conclusions: Tumor voxel (SUV 0 , DRM) provides an intratumoral prognostic map to target tumor locoregional-resistant regions. The corresponding TVCP or DPF provides a quantitative objective to optimize the intratumoral dose distribution for the individuals. The adaptive DPbN with FDG-PET/CT imaging feedback is feasible to implement in clinics., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: To summarize important talking points from a 2016 symposium focusing on real-world challenges to advancing precision medicine in radiation oncology, and to help radiation oncologists navigate the practical challenges of precision, radiation oncology., Methods and Materials: The American Society for Radiation Oncology, American Association of Physicists in Medicine, and National Cancer Institute cosponsored a meeting on precision medicine in radiation oncology. In June 2016 numerous scientists, clinicians, and physicists convened at the National Institutes of Health to discuss challenges and future directions toward personalized radiation therapy. Various breakout sessions were held to discuss particular components and approaches to the implementation of personalized radiation oncology. This article summarizes the genomically guided radiation therapy breakout session., Results: A summary of existing genomic data enabling personalized radiation therapy, ongoing clinical trials, current challenges, and future directions was collected. The group attempted to provide both a current overview of data that radiation oncologists could use to personalize therapy, along with data that are anticipated in the coming years. It seems apparent from the provided review that a considerable opportunity exists to truly bring genomically guided radiation therapy into clinical reality., Conclusions: Genomically guided radiation therapy is a necessity that must be embraced in the coming years. Incorporating these data into treatment recommendations will provide radiation oncologists with a substantial opportunity to improve outcomes for numerous cancer patients. More research focused on this topic is needed to bring genomic signatures into routine standard of care., (Published by Elsevier Inc.)

Purpose: To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts., Methods and Materials: Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2 Gy/d for a total dose of 20 Gy using a 5 days on, 2 days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40 Gy in 4 weeks using the same 2-Gy/d regimens., Results: Over the 2 weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31 ± 0.02 mm(3)/d and 0.55 ± 0.04 mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5% ± 3%), hypoxia (10% ± 3.5%), vascular endothelial growth factor (2.3% ± 1%), and stromal-derived factor-1α (2.5% ± 1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8% ± 2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6% ± 0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT., Conclusions: Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To assess the efficacy of 3-week schedules of low-dose pulsed radiation treatment (PRT) and standard radiation therapy (SRT), with concurrent cisplatin (CDDP) in a head and neck squamous cell carcinoma xenograft model., Methods and Materials: Subcutaneous UT-SCC-14 tumors were established in athymic NIH III HO female mice. A total of 30 Gy was administered as 2 Gy/d, 5 d/wk for 3 weeks, either by PRT (10 × 0.2 Gy/d, with a 3-minute break between each 0.2-Gy dose) or SRT (2 Gy/d, uninterrupted delivery) in combination with concurrent 2 mg/kg CDDP 3 times per week in the final 2 weeks of radiation therapy. Treatment-induced growth delays were defined from twice-weekly tumor volume measurements. Tumor hypoxia was assessed by (18)F-fluoromisonidazole positron emission tomography imaging, and calculated maximum standardized uptake values compared with tumor histology. Tumor vessel density and hypoxia were measured by quantitative immunohistochemistry. Normal tissues effects were evaluated in gut and skin., Results: Untreated tumors grew to 1000 mm(3) in 25.4 days (±1.2), compared with delays of 62.3 days (±3.5) for SRT + CDDP and 80.2 days (±5.0) for PRT + CDDP. Time to reach 2× pretreatment volume ranged from 8.2 days (±1.8) for untreated tumors to 67.1 days (±4.7) after PRT + CDDP. Significant differences in tumor growth delay were observed for SRT versus SRT + CDDP (P=.04), PRT versus PRT + CDDP (P=.035), and SRT + CDDP versus PRT + CDDP (P=.033), and for survival between PRT versus PRT + CDDP (P=.017) and SRT + CDDP versus PRT + CDDP (P=.008). Differences in tumor hypoxia were evident by (18)F-fluoromisonidazole positron emission tomography imaging between SRT and PRT (P=.025), although not with concurrent CDDP. Tumor vessel density differed between SRT + CDDP and PRT + CDDP (P=.011). No differences in normal tissue parameters were seen., Conclusions: Concurrent CDDP was more effective in combination PRT than SRT at restricting tumor growth. Significant differences in tumor vascular density were evident between PRT and SRT, suggesting a preservation of vascular network with PRT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the efficacy of low-dose pulsed radiation therapy (PRT) in 2 head and neck squamous cell carcinoma (HNSCC) xenografts and to investigate the mechanism of action of PRT compared with standard radiation therapy (SRT)., Methods and Materials: Subcutaneous radiosensitive UT-SCC-14 and radioresistant UT-SCC-15 xenografts were established in athymic NIH III HO female mice. Tumors were irradiated with 2 Gy/day by continuous standard delivery (SRT: 2 Gy) or discontinuous low-dose pulsed delivery (PRT: 0.2 Gy × 10 with 3-min pulse interval) to total doses of 20 Gy (UT14) or 40 Gy (UT15) using a clinical 5-day on/2-day off schedule. Treatment response was assessed by changes in tumor volume, (18)F-fluorodeoxyglucose (FDG) (tumor metabolism), and (18)F-fluoromisonidazole (FMISO) (hypoxia) positron emission tomography (PET) imaging before, at midpoint, and after treatment. Tumor hypoxia using pimonidazole staining and vascular density (CD34 staining) were assessed by quantitative histopathology., Results: UT15 and UT14 tumors responded similarly in terms of growth delay to either SRT or PRT. When compared with UT14 tumors, UT15 tumors demonstrated significantly lower uptake of FDG at all time points after irradiation. UT14 tumors demonstrated higher levels of tumor hypoxia after SRT when compared with PRT as measured by (18)F-FMISO PET. By contrast, no differences were seen in (18)F-FMISO PET imaging between SRT and PRT for UT15 tumors. Histologic analysis of pimonidazole staining mimicked the (18)F-FMISO PET imaging data, showing an increase in hypoxia in SRT-treated UT14 tumors but not PRT-treated tumors., Conclusions: Differences in (18)F-FMISO uptake for UT14 tumors after radiation therapy between PRT and SRT were measurable despite the similar tumor growth delay responses. In UT15 tumors, both SRT and PRT were equally effective at reducing tumor hypoxia to a significant level as measured by (18)F-FMISO and pimonidazole., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To investigate whether radiation treatment influences the expression of glucose metabolism genes and compromises the potential use of (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) as a tool to monitor the early response of head and neck cancer xenografts to radiation therapy (RT)., Methods and Materials: Low passage head and neck squamous cancer cells (UT14) were injected to the flanks of female nu/nu mice to generate xenografts. After tumors reached a size of 500 mm(3) they were treated with either sham RT or 15 Gy in 1 fraction. At different time points, days 3, 9, and 16 for controls and days 4, 7, 12, 21, 30, and 40 after irradiation, 2 to 3 mice were assessed with dynamic FDG-PET acquisition over 2 hours. Immediately after the FDG-PET the tumors were harvested for global gene expression analysis and immunohistochemical evaluation of GLUT1 and HK2. Different analytic parameters were used to process the dynamic PET data., Results: Radiation had no effect on key genes involved in FDG uptake and metabolism but did alter other genes in the HIF1α and glucose transport-related pathways. In contrast to the lack of effect on gene expression, changes in the protein expression patterns of the key genes GLUT1/SLC2A1 and HK2 were observed after radiation treatment. The changes in GLUT1 protein expression showed some correlation with dynamic FDG-PET parameters, such as the kinetic index., Conclusion: (18)F-fluorodeoxyglucose positron emission tomography changes after RT would seem to represent an altered metabolic state and not a direct effect on the key genes regulating FDG uptake and metabolism., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To examine the prognostic significance of c-Met expression in relation to p16 and epidermal growth factor receptor (EGFR) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with definitive concurrent chemoradiation., Methods and Materials: Archival tissue from 107 HNSCC patients treated with chemoradiation was retrieved, and a tissue microarray was assembled. Immunohistochemical staining of c-Met, p16, and EGFR was performed. c-Met expression was correlated with p16, EGFR, clinical characteristics, and clinical endpoints including locoregional control (LRC), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS)., Results: Fifty-one percent of patients were positive for p16, and 53% were positive for EGFR. Both p16-negative (P≤.001) and EGFR-positive (P=.019) status predicted for worse DFS. Ninety-three percent of patients stained positive for c-Met. Patients were divided into low (0, 1, or 2+ intensity) or high (3+ intensity) c-Met expression. On univariate analysis, high c-Met expression predicted for worse LRC (hazard ratio [HR] 2.27; 95% CI, 1.08-4.77; P=.031), DM (HR 4.41; 95% CI, 1.56-12.45; P=.005), DFS (HR 3.00; 95% CI, 1.68-5.38; P<.001), and OS (HR 4.35; 95% CI, 2.13-8.88; P<.001). On multivariate analysis, after adjustment for site, T stage, smoking history, and EGFR status, only high c-Met expression (P=.011) and negative p16 status (P=.003) predicted for worse DFS. High c-Met expression was predictive of worse DFS in both EGFR-positive (P=.032) and -negative (P=.008) patients. In the p16-negative patients, those with high c-Met expression had worse DFS (P=.036) than did those with low c-Met expression. c-Met expression was not associated with any outcome in the p16-positive patients., Conclusions: c-Met is expressed in the majority of locally advanced HNSCC cases, and high c-Met expression predicts for worse clinical outcomes. High c-Met expression predicted for worse DFS in p16-negative patients but not in p16-positive patients. c-Met predicted for worse outcome regardless of EGFR status., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model., Methods and Materials: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients., Results: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133(+) HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b(+) counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation., Conclusions: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the efficacy of pulsed low-dose radiation therapy (PLRT) combined with temozolomide (TMZ) as a novel treatment approach for radioresistant glioblastoma multiforme (GBM) in a murine model., Methods and Materials: Orthotopic U87MG hGBM tumors were established in Nu-Foxn1(nu) mice and imaged weekly using a small-animal micropositron emission tomography (PET)/computed tomography (CT) system. Tumor volume was determined from contrast-enhanced microCT images and tumor metabolic activity (SUVmax) from the F18-FDG microPET scan. Tumors were irradiated 7 to 10 days after implantation with a total dose of 14 Gy in 7 consecutive days. The daily treatment was given as a single continuous 2-Gy dose (RT) or 10 pulses of 0.2 Gy using an interpulse interval of 3 minutes (PLRT). TMZ (10 mg/kg) was given daily by oral gavage 1 hour before RT. Tumor vascularity and normal brain damage were assessed by immunohistochemistry., Results: Radiation therapy with TMZ resulted in a significant 3- to 4-week tumor growth delay compared with controls, with PLRT+TMZ the most effective. PLRT+TMZ resulted in a larger decline in SUVmax than RT+TMZ. Significant differences in survival were evident. Treatment after PLRT+TMZ was associated with increased vascularization compared with RT+TMZ. Significantly fewer degenerating neurons were seen in normal brain after PLRT+TMZ compared with RT+TMZ., Conclusions: PLRT+TMZ produced superior tumor growth delay and less normal brain damage when compared with RT+TMZ. The differential effect of PLRT on vascularization may confirm new treatment avenues for GBM., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: To investigate the metabolic information provided by (18)F-fluorodeoxyglucose-positron emission tomography (FDG-PET) during the early response of head-and-neck squamous cell carcinoma (HNSCC) xenografts to radiotherapy (RT)., Methods and Materials: Low-passage HNSCC cells (UT14) were injected into the rear flanks of female nu/nu mice to generate xenografts. After tumors grew to 400-500 mm(3), they were treated with either 15 Gy in one fraction (n = 18) or sham RT (n = 12). At various time points after treatment, tumors were assessed with 2-h dynamic FDG-PET and immediately harvested for direct histological correlation. Different analytical parameters were used to process the dynamic PET data: kinetic index (Ki), standard uptake value (SUV), sensitivity factor (SF), and retention index (RI). Tumor growth was assessed using the specific growth rate (SGR) and correlated with PET parameters using the Pearson correlation coefficient (r). Receiver operating characteristic (ROC) and the area under the ROC curve (AUC) were used to test PET parameters for their ability to predict for radiation necrosis and radiation change., Results: Tumor growth was arrested for the first 20 days after RT and recovered thereafter. Histologically, radiation change was observed in the peripheral regions of tumors between days 7 and 23 after RT, and radiation necrosis were observed in the central regions of tumors between days 7 and 40. Ki provided the best correlation with SGR (r = 0.51) and was the optimal parameter to predict for early radiation necrosis (AUC = 0.804, p = 0.07). SUV(30 min) was the strongest predictor for late radiation necrosis (AUC = 0.959, p = 0.004). Both RI(30-60 min) and SF(12-70 min) were very accurate in predicting for radiation change (AUC = 0.891 and 0.875, p = 0.009 and 0.01, respectively)., Conclusions: Dynamic FDG-PET analysis (such as Ki or SF) may provide informative assessment of early radiation necrosis or radiation change of HNSCC xenografts after RT., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging., Methods: Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CT (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields., Results: Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 × 10(6) cells produced a 50- to 70-mm(3) tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004)., Conclusion: This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To examine the combination of radiation and the multikinase inhibitor sorafenib in human colorectal cancer cell lines and xenografts., Methods and Materials: HT29 and SW48 colorectal cancer cells were studied in vitro using MTT assays to establish the optimal timing of radiation and sorafenib. This optimal timing was then investigated in clonogenic survival assays. Xenografts were established, and the effect of a 3-week schedule of daily radiation and sorafenib was studied by growth delay., Results: Sorafenib predominantly had minimal effects on cell growth or radiation response in MTT growth assays, though growth inhibition was significantly enhanced in HT29 cells when sorafenib was administered after radiation. The highest dose of sorafenib altered the alpha component of the cell survival curve in clonogenic assays. The combination of radiation and sorafenib was synergistic in SW48 xenografts, with a mean time to threshold tumor size of 11.4 +/- 1.0 days, 37.0 +/- 9.5 days, 15.5 +/- 3.2 days, and 98.0 +/- 11.7 days in the control, radiation, sorafenib, and combined treatment group, respectively. The effect on HT29 tumors was additive, with mean time to threshold volume of 12.6 +/- 1.1 days, 61.0 +/- 4.3 days, 42.6 +/- 11.7 days, and 100.2 +/- 12.4 days., Conclusions: Sorafenib had little effect on radiation response in vitro but was highly effective when combined with radiation in vivo, suggesting that inhibition of proliferation and interference with angiogenesis may be the basis for the interaction., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Purpose: An association between low-dose hyper-radiosensitivity (HRS) and the "early" G2/M checkpoint has been established. An improved molecular understanding of the temporal dynamics of this relationship is needed before clinical translation can be considered. This study was conducted to characterize the dose response of the early G2/M checkpoint and then determine whether low-dose radiation sensitivity could be increased by synchronization or chemical inhibition of the cell cycle., Methods and Materials: Two related cell lines with disparate HRS status were used (MR4 and 3.7 cells). A double-thymidine block technique was developed to enrich the G2-phase population. Clonogenic cell survival, radiation-induced G2-phase cell cycle arrest, and deoxyribonucleic acid double-strand break repair were measured in the presence and absence of inhibitors to G2-phase checkpoint proteins., Results: For MR4 cells, the dose required to overcome the HRS response (approximately 0.2 Gy) corresponded with that needed for the activation of the early G2/M checkpoint. As hypothesized, enriching the number of G2-phase cells in the population resulted in an enhanced HRS response, because a greater proportion of radiation-damaged cells evaded the early G2/M checkpoint and entered mitosis with unrepaired deoxyribonucleic acid double-strand breaks. Likewise, abrogation of the checkpoint by inhibition of Chk1 and Chk2 also increased low-dose radiosensitivity. These effects were not evident in 3.7 cells., Conclusions: The data confirm that HRS is linked to the early G2/M checkpoint through the damage response of G2-phase cells. Low-dose radiosensitivity could be increased by manipulating the transition of radiation-damaged G2-phase cells into mitosis. This provides a rationale for combining low-dose radiation therapy with chemical synchronization techniques to improve increased radiosensitivity., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines., Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity was assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing gammaH2AX., Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2 microg/mL], Trinovin [10 microg/mL], and Prostate Rx [50 microg/mL]). However, both Trinovin (10 microg/mL) and Prostate Rx (6 microg/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair., Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G(2)-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance., Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 microg/mL chloroquine, 15 microM genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eight cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G(2)-phase cells entering mitosis, using histone H3 phosphorylation analysis., Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G(2)-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range., Conclusion: Overcoming HRS is reliant on the function of the early G(2)-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.

Purpose: To investigate the localization and distribution of cytochrome P450 CYP1B1 protein expression in patients diagnosed with prostate carcinoma compared to those with bladder carcinoma. To validate CYP1B1 as a molecular target for the development of selective cancer therapeutics for use in combination with radiation., Methods and Materials: Prostatectomy specimens (n = 33) of moderate Gleason grade (3 + 3 and 3 + 4) were analyzed immunohistochemically for CYP1B1 protein expression using a specific monoclonal antibody for the enzyme. The intensity of CYP1B1 staining was assessed both semiquantitatively using visual scoring and quantitatively by spectral imaging microscopy using reference spectra and compared with bladder carcinoma (n = 22)., Results: CYP1B1 protein expression was present in 75% of prostate carcinomas (n = 27) compared to 100% of bladder carcinomas (n = 22). In both cases, CYP1B1 protein expression was heterogeneous and localized in the cytoplasm of the tumor cells but absent from the surrounding stromal tissue. CYP1B1 was also detected in premalignant prostatic intraepithelial neoplasia (n = 2, 100%), as well as noncancerous tissues, including benign prostatic hyperplasia (n = 27, 82%), metaplastic prostatic urothelium (n = 8, 100%), and hyperplastic prostatic urothelium (n = 14, 100%). Higher CYP1B1 protein expression in bladder vs. prostate carcinoma was confirmed by their corresponding average normalized absorbances (+/- standard deviation), measured as 1.40 +/- 0.44 and 0.55 +/- 0.09, respectively. Overall CYP1B1 staining intensity in prostate carcinoma was similar to that in prostatic intraepithelial neoplasia, benign prostatic hyperplasia, and hyper-/metaplastic urothelial tissue. No CYP1B1 was detected in normal prostate tissue., Conclusions: CYP1B1 is overexpressed in prostate carcinoma at a high frequency and is also detectable in the associated premalignant and hyperplastic tissue, implicating a possible link with malignant progression and CYP1B1 as a suitable target for therapy. Spectral imaging microscopy has highlighted differences in CYP1B1 protein expression between different cancers.

Purpose: Tumor cell repopulation is still considered to be a major cause of failure in radiotherapy. In this study, we investigated the influence of cell kinetic parameters on the outcome of patients treated in a randomized trial of accelerated fractionation, with or without mitomycin C, vs. conventional fractionation., Methods and Materials: Sixty-two patients were studied using administration of bromodeoxyuridine (BrdUrd), and cell kinetic parameters were measured using flow cytometry. The patients were treated with either 70 Gy for 7 weeks or 55.3 Gy for 17 continuous days (V-CHART) with or without 20 mg/m(2) mitomycin C on day 5., Results: The potential doubling time (Tpot) and labeling index (LI) failed to provide any prognostic information with regard to local control or survival. However, the duration of the S phase (Ts) revealed patients whose tumors had a long Ts had significantly worse local control (p = 0.028) and survival (p = 0.034) irrespective of treatment. A similar trend was evident within the different treatment arms particularly associated with overall survival., Conclusions: The Ts values of head-and-neck squamous cell cancers provided prognostic information that predicted clinical outcome irrespective of treatment schedule in this study. This neglected parameter of the Tpot method might provide information related to redistribution of cells during fractionated radiotherapy.

Purpose: The role of bcl-2 overexpression in cancer presents a paradox. In some tumor types, it is associated with favorable outcome, whereas in others the reverse is true. The purpose of this study was to explore the influence of bcl-2 in a large series of head and neck cancer patients treated in the CHART randomized trial., Methods and Materials: Histologic material was obtained from 400 patients; bcl-2 expression was assessed by immunohistochemistry as either positive or negative cytoplasmic staining., Results: Positivity of bcl-2 was recorded in 12.8% (9.5-16.5%, 95% confidence limits) of tumors. There were significant differences in positive tumors within different sites with nasopharynx showing the highest incidence (46.2%). A multivariate logistic regression analysis showed that bcl-2 was strongly associated with histologic dedifferentiation, as well as increasing N stage and female gender. In univariate analyses, bcl-2 positive patients had a lower locoregional relapse rate (RR 0.57, p = 0.02) and improved survival (RR 0.49, p = 0.004) compared to bcl-2 negative patients; this became more significant in multivariate analysis., Conclusion: These data demonstrate that bcl-2 overexpression is a marker of what is considered to be more advanced and aggressive disease yet it is associated with a more favorable outcome irrespective of the treatment schedule.

Purpose: The aim of the study was to present the updated Institut Gustave Roussy experience of the predictive value of three biological parameters in patients with squamous cell carcinoma of the Head and Neck (HNSCC) treated with radiation therapy., Methods and Materials: Three parameters have been investigated independently: tumor cell kinetics (TS, Tpot and LI), oxygen tension measurements (PO2) and intrinsic radiosensitivity (SF2Gy)., Results: No relationship has been found between local-regional control and Tpot or LI in a series of 74 patients. Our data also support that the surviving fraction at 2 Gy, (SF2) was unlikely to predict the clinical outcome in a series of 92 patients. Differences in PO2 measurements have been observed between tumors, and tumor oxygenation was lower than that of normal tissue for the majority of patients. However PO2 measurements did not predict clinical outcome, but further investigations are needed to draw definitive conclusions, given the limited number of patients entered in our study (35 patients). In addition, we were able to measure the three parameters in 10 patients showing no correlation between PO2, SF2 and Tpot., Conclusions: The method used to evaluate Tpot and SF2 did not provide clinically relevant predictive parameters for this type of cancer. Further investigations are needed to assess the predictive value of PO2 measurements and of new biological parameters in a multiparametric approach, taking into account other possible clinical and biological confounding factors.

Purpose: The goal of this study was to investigate the clinical utility of pretreatment measurements of tumor cell kinetics to predict the outcome of patients with squamous cell carcinoma of the head and neck receiving conventional radiotherapy., Methods and Materials: All patients received between 64 and 70 Gy as 2 Gy fractions, five fractions per week. Cell kinetics were assayed rapidly and quantitatively using flow cytometric evaluation of iododeoxyuridine (IdUrd) incorporation, in vivo, from a biopsy removed several hours after the administration of the DNA precursor to the patient prior to the start of treatment., Results: The measured proliferation parameters were not related to the clinicopathological features of the tumors, emphasizing the independent nature of these parameters. In univariate analysis, nodal involvement was the most important clinical feature of the tumors related to local control followed by Tpot, DNA aneuploidy, and attainment of complete regression at 6 weeks. Of these only Tpot and nodal status maintained significance in multivariate analysis, with respect to loco-regional control. In subgroup analysis, Tpot was able to stratify patients into high or low rate of loco-regional control in node negative patients, in aneuploid tumors and in patients who did achieve complete regression at 6 weeks. For cause specific survival, N-stage was the only parameter that significantly discriminated the prognosis in these patients., Conclusions: The conclusion of this study is that Tpot provides clinically important information that can predict patients with a low probability of achieving long-term local control with conventional fractionation. Further improvements to the methodology to address the shortcomings of analyzing diploid tumors may increase the predictive power of the measurement.

Purpose: To assess the prognostic value of the pretreatment potential doubling time (Tpot) in carcinoma of the uterine cervix, relative to other established clinical factors., Methods and Materials: Fifty-two patients with cervical cancer were studied prospectively from March 1991 to October 1993. Pretreatment evaluation included examination under anesthesia and tumor biopsy 6 h following the intravenous administration of bromodeoxyuridine (200 mg). Tpot was determined by deriving the labeling index (LI) and S-phase synthesis time (Ts) using flow cytometry. Six patients were not evaluable and excluded. The remaining 46 patients (average age 55 years) were treated uniformly with radical radiation therapy. There were 39 squamous carcinomas and 7 adenocarcinomas. Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stages were: Ib and IIa, 12 patients; IIb, 18 patients; III and IV, 16 patients. The median external beam dose was 50 Gy (range, 45-52.8 Gy) delivered in 25 fractions. The median intracavitary dose was 40 Gy (range. 25.5-40 Gy) delivered with a single line source to a point 2 cm lateral of the midline, with a mean dose rate of 0.71 Gy/h. The median overall treatment time was 45 days (range, 34-73 days). As of July 31, 1994, 12 patients had died of disease, and the average follow-up for alive patients was 1.4 years (range, 0.5-3.3 years)., Results: There were 27 tumors with diploid deoxyribonucleic acid (DNA) content and 19 tumors were aneuploid. The median and mean Tpot for the 46 patients were 5.5 and 6.6 days, respectively [range, 2.0-25.6 days; coefficient of variation (CV), 74%]. For 25 patients where Tpot measurements were performed at two separate laboratories, there was a fair correlation (r = 0.74), but systematic differences were detected suggesting that the lack of agreement was not simply due to intratumoral variation. To date, 30 patients remained disease free, while 8 patients had pelvic failure and 9 patients developed distant metastases as the first failure site (1 patient developed both at the same time). In univariate analysis, the only significant prognostic factor for disease-free survival was tumor size (p = 0.004). A short Tpot (or high LI) and long overall treatment time (OTT) were weakly associated with poorer disease-free survival, although not statistically significant (1/Tpot, p = 0.14; LI, p = 0.23; OTT, p = 0.04). Age, FIGO stage, hemoglobin level, S-phase fraction, DNA ploidy, and Ts were not associated with disease-free survival. Multiple regression analysis was not performed because of the relatively small number of patients and short follow-up., Conclusions: Tpot values determined with current techniques by different laboratories cannot be used interchangeably for the purpose of therapy decisions. Vigorous quality assurance and standardization of the laboratory procedures and analysis methods are important to reduce interlaboratory variation. In this uniformly treated group of patients with cancer of the uterine cervix, traditional clinical prognostic factors remain the most important. Preliminary data suggest that the flow cytometry-determined Tpot and labeling index predict for disease-free survival, although a larger number of patients with longer follow-up is required to assess the true prognostic significance of these assays and to determine if their effect is independent of other clinical factors.

Purpose: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed., Methods and Materials: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy gamma rays. Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd). At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions. The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically. The kidneys were also assessed histologically., Results: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 +/- 0.32% and 1.04 +/- 0.13%, respectively, were seen 4 weeks PI, significantly greater (P < 0.001) than the preirradiation value of 0.18 +/- 0.01%. The LI values then declined with time, but remained greater than those seen prior to irradiation. A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically. The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI. The irradiated kidney exhibited diffuse, progressive glomerular alterations. In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening. The contralateral unirradiated kidney appeared unchanged., Conclusion: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys. Renal irradiation results in significant alterations in glomerular and tubular cell proliferation and morphology within 2-4 weeks of irradiation; glomerular changes appear predominant.

The local tumor control achieved in patients treated in a pilot study of continuous, hyperfractionated, accelerated radiotherapy has been related to the tumor cell kinetics evaluated by in vivo administration of bromodeoxyuridine and flow cytometry. In 42 of 50 patients with advanced squamous cell carcinomas in the head and neck region it was possible to sample the primary tumor prior to treatment. In three further cases, involved regional nodes were studied: in the remaining five, tissue was obtained subsequently either from a local recurrence or from a distant metastasis. Successful cell kinetic measurements were made in 38 (90%) of the 42 primary tumors. The median values of the labelling index, the duration of the DNA synthetic phase and, thus, the potential doubling time for all primaries were 7.1%, 9.8 hr, and 3.9 days, respectively. Complete regression was achieved in 28 (74%) of the primary tumors and in 23 (61%) this was maintained to the time of observation for this report. There was no significant influence of any of the cell kinetic parameters upon the immediate or longer term local tumor control. This result is compatible with the overcoming of cellular repopulation by the acceleration of radiotherapy.

To evaluate the therapeutic potential of normobaric oxygen and carbogen as hypoxic-cell sensitizers, both radiosensitization in a mouse mammary carcinoma, mouse skin and kidneys, and the reduction in the proportion of hypoxic tumor cells were quantified in mice breathing air, oxygen, or carbogen. Local tumor control, acute skin reactions, reduced renal clearance, and hematocrit were used as assays. X rays as 10 fractions in 5 days were given to skin and tumors and 10F/12 days to kidneys. In the tumor study, the pre-irradiation breathing time was varied from 2 to 20 min. Hypoxic cells, before and during a 10F/5 day schedule, were quantified using a 2-nitroimidazole with a theophylline side chain. Bioreductively reduced metabolites of this probe were localized in hypoxic cells that were then stained using an immunofluorescent technique and analyzed by flow cytometry. The fraction of cells with high fluorescence intensity was 19% in air, 9% in oxygen, and 3% in carbogen-breathing mice. For all three gases, hypoxia-dependent binding was similar in non-irradiated tumors and those treated with four or nine fractions. Both gases significantly enhanced tumor radiosensitivity (ER = 1.3 to 1.6) and carbogen was slightly more effective than oxygen. With carbogen, maximum sensitization was observed with a 5 min pre-irradiation breathing interval. With oxygen, pre-irradiation breathing times of 2-20 min gave similar sensitization. In skin an enhancement ratio of 1.2 was observed, whereas enhancement ratios for both renal endpoints were significantly lower (1.0 to 1.07). Relative to both tissues, there was therefore a substantial therapeutic gain by irradiating CaNT tumors under both gases, especially with carbogen.